The use of fluorophores for identifying or localizing structures within objects is of considerable importance in diverse fields and in biological/medical applications in particular. Optical fluorescence imaging of turbid media such as biological tissue, is primarily achieved with Continuous Wave (CW) methods. Typically a light source is employed to illuminate the object of interest, e.g. a mouse in vivo, and the emitted CW fluorescence intensity signal from the fluorophore is measured directly with a camera. Systems in which the fluorescence from small animals comprising bioluminescent molecules can be measured have been described. See for example, U.S. Pat. Nos. 6,775,567 and 5,650,135.
For applications such as diagnosis and pharmacological studies, it is often desirable to determine the concentration and depth of the fluorophore. However, to assume that the direct CW fluorescence intensity signal is proportional to the fluorophore concentration can be misleading since the depth of the fluorophore will also impact the CW fluorescence intensity signal. In fact, given a single CW source and CW detector measurement it is impossible to decouple fluorophore concentration and depth.
To address this problem of decoupling depth and concentration in CW measurements, some researchers are exploring CW tomography, in order to reconstruct a three-dimensional distribution of the fluorophore concentration in the object, thereby attempting to account for the depth of the fluorophore. However, these tomographic approaches require multiple source-detector pair measurements from many angles, combined with complex, computer intensive inversion algorithms. Furthermore, CW tomography requires an assumption about the scattering coefficient of the object, since CW can neither decouple the intrinsic absorption and scattering of the object.
The most complete description of photon migration in turbid media is provided by Time Domain (TD) optical methods which have previously been used to decouple the attenuation coefficient, given from CW intensity measurements, into the underlying absorption and scattering coefficients. However TD methods have not been applied to obtain depth and concentration of fluorophores.